Outline

The severe acute respiratory syndrome associated coronavirus (SARS-CoV) causes severe pneumonia with fatal outcome in approximately 10% of the patients. The SARS-CoV is not closely related to other coronaviruses, but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARS-CoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T and B-cell lines. Infection of the hepatoma cell line Huh-7 was also observed with replication competent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S bearing pseudotypes, indicating that S mediated entry requires low pH. Infection by SARS-CoV S pseudotypes but not by VSV-G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera from SARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV infected patients. The viral attachment factors DC-SIGN and DC-SIGNR enhanced S driven infection of cell lines, providing evidence that expression of these lectins might enhance SARS-CoV infection of certain target cells in vivo. Finally, S mediated entry into susceptible cells was enhanced by the recently identified receptor ACE2 and susceptibility to pseudotype infection correlated with ACE2 expression, suggesting that ACE2 might be the only receptor for SARS-CoV. Our results show that viral pseudotyping can be employed for the analysis of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue, depends on ACE2 expression and can be inhibited by the humoral immune response in infected patients.